WO2021185851A1 - Vaccine compositions for hiv prevention and treatment - Google Patents

Abstract

The inventors used a reverse vaccinology approach based on the Fab conformational epitopes to generate a (poly) peptide that is useful in therapeutic approaches against HIV infection. Accordingly, the invention relates to a novel peptide relating to HIV is disclosed and therapeutic compositions containing such peptide are discloseda. The compound and compositions of the invention are of use in prevention and treatment of HIV, and in particular for inducing mucosal immunity to HIV.

Description

VACCINE COMPOSITIONS FOR HIV PREVENTION AND TREATMENT

FIELD OF THE INVENTION

The invention is in the infection field. More particularly, the invention relates to novel (poly) peptides relating to HIV are disclosed, as are vaccine compositions containing such (poly) peptides. The compounds and compositions of the invention are of use in prevention and treatment of HIV, and in particular for inducing mucosal immunity to HIV. BACKGROUND OF THE INVENTION

HIV-1 infection is mainly initiated at mucosal sites during sexual intercourses, and IgA can efficiently prevent HIV-1 infection and mucosal reservoir establishment. The inventor and others have previously shown that mucosal IgAs, specific for HIV-1 envelope gp41-subunit conserved epitopes, naturally raise in 1% of individuals upon sexual exposure to HIV-1 who remain persistently uninfected. These individuals, referred to as Exposed SeroNegatives (ESN), remain IgG seronegative but raise gp41 -specific mucosal IgA that constitute a main correlate of protection (reviewed in [1] [2] [5]). In vitro, ESN IgA are able to inhibit HIV-1 mucosal penetration by transcytosis across epithelial barriers and to neutralize CD4⁺T-cell infection against Clade B HIV-1 [2] [6] [7] These IgA, detected mainly in vaginal washes and urine, recognize the conserved regions of the transmembrane subunit gp41 of HIV-1 envelope [6] [2, 7] Remarkably, at the molecular level, ESN IgAs are affinity matured with extensive somatic hypermutations, and have extended heavy-chain third complementary determining regions, (CDRH3) [2], characteristics similar to those found for IgGs in natural HIV-1 infection and that increase breadth and potency in vitro [8] [9] [10] Compared to HIV-1 infected patient IgGs, which unfortunately develop too late (several years) to prevent infection, mucosal ESN IgAs raise early after HIV-1 exposure and offer protection from infection. The presence of ESN protective gp41 -specific IgAs at mucosal level evidences an efficient “natural” vaccination. Furthermore, this IgA-driven immune response in ESN questions about the protective mechanisms of a humoral response involving only mucosal IgA, and, at the molecular level, about the participation of the isotype specific constant regions of the antibody in ESN IgA efficacy.

Antibodies (Ab) have been historically described as being composed of two distinct structurally independent domains, namely the variable regions, forming the paratope, responsible for antigen binding, and the heavy chain constant regions mediating effector functions cells such as antibody dependent cell cytotoxicity (ADCC) and antibody dependent phagocytosis (ADCP). Recent works including those of the inventor [11], revealed that the Ab isotype carried by the Ab heavy chain constant region (CH) influences the paratope structure and in turn the antigen recognition. Modulation of antigen recognition by CH has been reported for IgM, IgG, IgE and IgA, using a variety of biochemical and biophysical techniques. Hence, by isotype switching a broadly neutralizing 2F5 IgGl specific for gp41 derived from the blood of an HIV- 1 -infected patient [13] into a IgA2, the inventor previously showed that the CH influences affinity and epitope specificity, and in turn, antiviral functions [11] Changes induced by isotype switching occur most likely by modulation of the paratope conformation due to non-local [14] and even allosteric effects that modify Ab- antigen interaction [15], antigen binding affecting the constant region and vice versa, as hypothesized by Ofran and col. [16] The CH contains three domains, namely CHI, CH2 and CH3. CH3 and CH2 participate in Fc- receptor binding on effector cells to mediate ADCC or ADCP whereas the CHI domain, separated from the two others by the hinge region, is part of the Fab region of the Ab and closer to the paratope formed by the variable heavy (VH) and light (VL) chains. Which CH domain(s) participates in modulation of Ab specificity and affinity remains unclear and whether the CHI itself, due to its proximity to the VH and VL regions, plays a determinant role in Ab affinity and functions has not been studied. Furthermore, the role of constant regions has only been evaluated by comparing blood derived antibodies expressing identical V regions in different heavy chain constant context and thus addressing a blood raised B-cell epitope. However, mucosal immunity is highly compartmentalized [17] resulting in the induction of mucosal antibody recognizing B cell epitope that differ from systemic one. Whether the antibody isoptype also affects Ab specificity and affinity and even function of mucosal Abs in a similar manner than blood derived Abs remains unknown.

Most of the vaccine approaches against HIV-I have targeted the viral envelope (Env) glycoproteins because they are the major surface antigens expressed on virions and by HIV-I infected cells. The native HIV-I envelope glycoprotein is a heterotrimer containing three gpl20 proteins non-covalently associated with three gp41 glycoproteins.

The three most potent HIV-I neutralizing antibodies yet identified, bl2, 2G12 and 2F5, have a high affinity for the native trimer. There is increasing effort to develop recombinant proteins as candidate vaccines that are better antigenic mimics than the native envelope glycoprotein complex such as the gpl20/gp41. However, due to numerous molecular homologies between the gpl20/gp41 and molecules of the immune system, many potential cross-reactivities events may occur leading to possible harmful autoimmune phenomena.

Various strategies have been described in order to dampen those cross- reactivities for obtaining anti-HIV vaccines with no or less cross reactivities with human proteins as the introduction of mutations and/or deletions in various part of the gp41 proteins as described in US 6,455,265 and WO 2005/010033.

However there is still a need for a vaccine that allows for inducing a versatile immune response against HIV infection, and in particular HIV-type 1 infection. There is also a need for the development of non-clade B vaccines, such as, for example, clade A and C strains.

There is also a need for the development of a vaccine with broad inhibitory spectrum allowing for cross-clade inhibition.

There is a need for a vaccine allowing to induce a humoral and/or cellular immune response against HIV infection.

There is a need for a vaccine allowing to induce an immune response against HIV infection at the mucosal surface level and at the blood level.

There is a need for a vaccine suitable for inducing mucosal IgA antibodies and systemic IgG antibodies.

It is an object of the invention to satisfy to all those above-mentioned needs. DESCRIPTION OF THE INVENTION

The inventor took advantage of two ESN mucosal anti-HIV-1 Fab-IgAl (FabA) previously characterized [2], that were isotype switched into Fab-IgGl (FabG). Each FabA and its respective isotype switched FabG were compared for HIV-1 envelope binding, epitope recognition, and anti- viral activities against the main HIV-1 strains present worldwide, namely HIV-1 of Clades B, A and C, and their conformational epitopes on gp41 were determined using in silico analyses. These comparative analyses result in a molecular understanding of the recognition of different epitopes on HIV-1 envelope, depending on the isotype in correlation with different affinities and antiviral functions. Based on these results, the inventors used a reverse vaccinology approach based on the Fab conformational epitopes to generate a (poly) peptide that is useful in therapeutic approaches against HIV infection.

Thus, in one aspect the invention provides a peptide comprising or consisting of the consecutive sequence of amino acids (SEQ IDN°1): LWNWFDISAASI

The peptide is useful in preventive and curative treatments of HIV infections.

In a first aspect, the invention provides a therapeutic composition comprising a peptide of the invention, wherein the peptide is used for its direct neutralizing properties against an HIV infection, namely by clade A, B and C virus.

According to this aspect, the peptide is used preferably systemically, as an antiviral agent.

In a second aspect, the invention provides a therapeutic composition comprising a peptide of the invention, wherein the peptide is used for its antigenic properties for inducing a protective immunity against an HIV infection.

Also provided are nucleic acids encoding the peptides of the invention, vectors comprising said nucleic acids and host cells comprising said vectors and nucleic acids.

Also provided is a composition comprising a peptide, or nucleic acid of the invention. In a preferred embodiment, said composition is a vaccine composition. In some embodiments, said composition is suitable for mucosal administration. For example, said composition may be suitable for administration to the nasal, rectal or vaginal mucosa. In some embodiments, said vaccine composition additionally comprises one or more adjuvants.

Also provided is a peptide, nucleic acid or composition of the invention for use in the preventive or therapeutic treatment of HIV or AIDS. The composition may be for administration to a mucosal surface, or any other administrative route such as those described below. The composition may be for administration in a prime-boost regimen such as those described below, such as a regimen comprising administration of full-length HIV-1 gp41.

Also provided is a peptide, nucleic acid or composition of the invention for use in a method of inducing a mucosal immune response to HIV-1 n some embodiments, said method comprises administering said peptide, nucleic acid or composition to a subject, in particular a subject in need thereof. The peptide, nucleic acid or composition may be administered to a mucosal surface of a subject, for example the nasal, rectal or vaginal mucosa. Various administration regimens are described herein and encompassed within the scope of the invention.

Also provided is a peptide, nucleic acid or composition of the invention for use in a method of inducing a mixed mucosal IgGl and IgA2 response to HIV-1, the method comprising administration of at least a peptide comprising the peptidic sequence of the invention or nucleic acids encoding said peptides, or a composition comprising said peptides or nucleic acids.

Also provided is the use of a peptide, nucleic acid or composition of the invention for use in the preparation of a medicament for the preventive treatment of HIV infection, in particular HIV- 1 infection, or AIDS.

Also provided is a method of prevention or treatment of HIV infection, in particular HIV-1 infection, comprising administration of a composition of the invention to an individual in need thereof. In some embodiments, said composition is administered to a mucosal surface of a subject, for example the nasal, rectal or vaginal mucosa. Various administration regimens are described herein and encompassed within the scope of the invention.

Also provided is a method of inducing a mucosal immune response to HIV-1, comprising prevention or treatment of HIV infection, comprising administration of a composition of the invention to an individual in need thereof. In some embodiments, said composition is administered to a mucosal surface of a subject, for example the nasal, rectal or vaginal mucosa. Various administration regimens are described herein.

Also provided is a method of inducing a mucosal IgA response to HIV-1, the method comprising administering at least one peptide as defined above or a nucleic acid encoding said peptide, or a composition comprising said peptide or nucleic acid, to an individual in need thereof, as described herein.

Also provided is a method of inducing a mucosal IgG response to HIV-1, the method comprising administering at least one peptide as defined above or a nucleic acid encoding said peptide, or a composition comprising said peptide or nucleic acid, to an individual in need thereof, as described herein.

Also provided is a method of inducing a mixed mucosal IgG and IgA response to HIV-1, the method comprising administering at least one peptide as defined above or nucleic acids encoding said peptides, or a composition comprising said peptide or nucleic acid to an individual in need thereof, as described herein. Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side- chains and the amino or carboxyl termini, it will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from natural posttxans!ational processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, araidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidyl inositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxy!ation, glycosylation, GPI anchor formation, hydroxy! ation, iodination, mefhylation, myristoyl ation, oxidation, proteolytic processing, phosphorylation, prenylation, racemizaiion, selenoyi ation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginyiation, and ubiquitination.

Variants of the peptide disclosed herein comprise peptides sequences of SEQ åDN°1 wherein 1 to 2 amino acids are substituted with other amino acids resulting in conservative substitutions. Exemplary conservative substitutions are listed below.

Ala (A) Val (V); Leu (L), lie (I)

Asn (N) Gin (Q); His (H); Lys (K)

Asp (D) Glu (E) lie (I) Leu (L); Val (V); Met (M); Ala (A)

Leu (L) Norleucine; He (I); Met (M); Ala (A); Phe (F)

Phe (F) Leu (L); Val (V); He (I); Ala (A); Tyr (Y)

Ser (S) Thr (T)

Trp (W) Tyr (Y); Phe (F)

The peptide vaccines of the invention may be formulated into a pharmaceutical form, preferably in combination with a pharmaceutically acceptable carrier. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions in all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intraarterial, intramuscular, subcutaneous, intratumoral and intrap eritoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in I ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, (see for example, Remington: The Science and Practice of Pharmacy, 21^st Edition, Lippincot and Williams, 2005). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The compositions disclosed herein may be formulated in a neutral or salt form. Pharmaceutically-acceptable salts, include the acid addition salts and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.

As used herein, ‘carrier’ includes, without limitation, solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, liposomes and virosomes such as those described in Bomsel et al (2011) Immunity 34: 269-280. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

The phrase ‘pharmaceutically-acceptable’ or ‘pharmacologically-acceptable’ refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human. The preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.

The vaccine composition will preferably contain an agent that enhances the protective efficacy of the vaccine, such as an adjuvant. Adjuvants include any compound or compounds that act to increase a protective immune response to the peptide antigen, thereby reducing the quantity of antigen necessary in the vaccine, and/or the frequency of administration necessary to generate a protective immune response. Adjuvants can include for example, emulsifiers; muramyl dipeptides; avridine; aqueous adjuvants such as aluminum hydroxide; oxygen-containing metal salts; chitosan-based adjuvants, and any of the various saponins, oils, and other substances known in the art, such as Ampfaigen, LPS, bacterial cell wall extracts, bacterial DNA, CpG sequences, synthetic oligonucleotides and combinations thereof (Schijns et al (2000) Curr. Opin. Immunol, 12:456), Mycohacterialplilei ( phlei) cell wall extract ( CWE) (U.S. Patent No. 4,744,984), M. phlei DNA (M-D A), and M-DNA-M phlei ceil wall complex (MCC), heat-labile enterotoxin (LT), cholera toxin (CT), cholera toxin B subunit (CTB). Compounds which can serve as emulsifiers include natural and synthetic emulsifying agents, as well as anionic, cationic and nonionic compounds. Oxygen-containing metal salts include salts of Al, K, Ca, Mg, Zn, Ba, Na, Li, B, Be, Fe, Si, Co, Cu, Ni, Ag, Au, and Cr which are sulphates, hydroxides, phosphates, nitrates, iodates, bromates, carbonates, hydrates, acetates, citrates, oxalates, and tartrates, and mixed forms thereof, including aluminium hydroxide, aluminium phosphate, aluminium sulphate, potassium aluminium sulphate, calcium phosphate, Maalox (mixture of aluminium hydroxide and magnesium hydroxide), beryllium hydroxide, zinc hydroxide, zinc carbonate, zinc chloride, and barium sulphateAmong the synthetic compounds, anionic emulsifying agents include, for example, the potassium, sodium and ammonium sails of lauric and oleic acid, the calcium., magnesium and aluminum salts of fatty acids, and organic sulfonates such as sodium lauryl sulfate. Synthetic cationic agents include, for example, cetyltrhethylammonlum bromide, while synthetic nonionic agents are exemplified by glycerylesters (e.g., glyceryl monostearate), polyoxyethylene glycol esters and ethers, and the sorbitan fatty acid esters (e.g., sorbitan monopalmitate) and their polyoxyethylene derivatives (e.g., polyoxyethylene sorbitan. monopalmitate). Natural emulsifying agents include acacia, gelatin, lecithin and cholesterol.

Other suitable adjuvants can be formed with an oil component, such as a single oil, a mixture of oils, a water-in-oil emulsion, or an oil-in- water emulsion. The oil can be a mineral oil, a vegetable oil, or an animal oil. .Mineral oils are liquid hydrocarbons obtained from petrolatum via a distillation technique, and are also referred to in the art as liquid paraffin, liquid petrolatum,, or white mineral oil. Suitable animal oils include, for example, cod liver oil, halibut oil, menhaden oil, orange roughy oil and shark liver oil, ail of which are available commercially. Suitable vegetable oils, include, for example, canola oii, almond oil, cottonseed oil, com oil, olive oil, peanut oil, safflower oii, sesame oil, soybean oil, and the like. Freund's Complete Adjuvant (FCA) and Freund's incomplete Adjuvant (FI A) are two common adjuvants that are commonly used in vaccme preparations, and are also suitable for use in the present invention. Both FCA and FIA are water-in-mineral oil emulsions; however, FCA also contains a killed Mycobacterium sp. Particularlay preferred adjuvants for mucosal vaccines include galactosyl ceramide (GalCer), as described in Lee et al (2011) Vaccine 29: 417-425.

Immunomodulatory cytokines can also be used in the vaccine compositions to enhance vaccine efficacy, for examplean adjuvant, Non-limiting examples of such cytokines include interferon alpha (IFN-a), interleukin-2 (IL-2), and granulocyte rnacrophage-colony stimulating factor (GM— CSF), or combinations thereof. GM-CSF is preferred.

When provided prophylactically, the immunogenic compositions of the invention are ideally administered to a subject in advance of HIV infection, or evidence of HIV infection, or in advance of any symptom due to AIDS, especially in high-risk subjects. The prophylactic administration of the immunogenic compositions can serve to provide protective immunity of a subject against HIV-1 infection or to prevent or attenuate the progression of AIDS in a subject already infected with HIV-1. When provided therapeutically, the immunogenic compositions can serve to ameliorate and treat AIDS symptoms and are advantageously used as soon after infection as possible, preferably before appearance of any symptoms of AIDS but may also be used at (or after) the onset of the disease symptoms.

Administration may be via a parenteral or non-parenteral route. Routes of administration will vary, naturally, with the location and nature of the disease, and include, e.g. intravenous, intrarterial, intradermal, transdermal, intramuscular, mucosal subcutaneous, percutaneous, intratracheal, intraperitoneal, perfusion and lavage. Preferably, administration is via a mucosal route, for example via a nasal, oral (via the mucosa of the digestive system), vaginal, buccal, rectal, sublingual, ocular, urinal, pulmonal or otolar (vie the ear) route.

For nasal administration, an exemplary formulation may be a nasal spray, lavage, drop or squirt system such as the Bidose Liquid from Aptar, Pfeffer Group or the Accuspray from Becton Dickinson (see Brandztaeg, 2011, AJRCCM, Bitter et al (2011) CurrProbl Dermatol 40: 20-35).

The treatments may include various ‘unit doses.’ A unit dose is defined as containing a predetermined-quantity of the therapeutic composition comprising a lentiviral vector of the present invention. The quantity to be administered, and the particular route and formulation, are within the skill of those in the clinical arts. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. A unit dose may contain at least 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0 or 50.0 mg of the active ingredient. Optionally, a unit dose contains less than 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0 or 50.0 mg of the active ingredient. In one embodiment, a unit dose contains from about 0.001 mg to about 50 mg of the active ingredient. In another embodiment a unit dose contains from about 1 mg to about 10 mg of active ingredient.

In one embodiment, the vaccine composition may be administered in a single daily dose, or the total daily dosage may be administered in divided doses, for example, two, three or four times daily. Furthermore, the vaccine composition may be administered in intranasal form via topical use of suitable intranasal vehicles, via transdermal routes, using those forms of transdermal skin patches well known to persons skilled in the art, by implantable pumps; or by any other suitable means of administration. To be administered in the form of a transdermal delivery system, for example, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.

The dosage regimen utilizing the vaccine composition is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound employed. A physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the disease or disorder that is being treated.

Vaccine administration may further comprise a prime-boost regimen. In these methods, one or more priming immunisations are followed by one or more boosting immunisations. The actual immunogenic composition can be the same or different for each immunisation and the type of immunogenic composition, the route, and formulation of the immunogens can also be varied. One useful prime-boost regimen provides for two priming immunisations, four weeks apart, followed by two boosting immunisations at 4 and 8 weeks after the last priming immunisation.

Such a regimen may comprise, for example, priming with full-length gp41 and boosting with one or more immunogenic peptides as disclosed herein. Either the prime or the bosst, or both, may be administered in the form of a DNA molecule encoding the peptide or polypeptide in question. Without being bound by theory, it is thought that this regimen might elicit gp41 cross-reactive antibodies targeted to the epitopes present in the priming immunogen. The designed protein fragments may be expressed in E. coli in order to prevent glycosylation and consequent epitope masking that might occur if expressed in a eukaryotic expression system. The use of E. coli to produce non-glycosylated versions of the invention may have contributed to the success of the approach.

Immunisation schedules (or regimens) are well known for animals (including humans) and can be readily determined for the particular subject and immunogenic composition. Hence, the immunogens can be administered one or more times to the subject. Preferably, there is a set time interval between separate administrations of the immunogenic composition. While this interval varies for every subject, typically it ranges from 10 days to several weeks, and is often 2, 4, 6 or 8 weeks. For humans, the interval is typically from 2 to 6 weeks. In a particularly advantageous embodiment of the present invention, the interval is longer, advantageously about 10 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks, 26 weeks, 28 weeks, 30 weeks, 32 weeks, 34 weeks, 36 weeks, 38 weeks, 40 weeks, 42 weeks, 44 weeks, 46 weeks, 48 weeks, 50 weeks, 52 weeks, 54 weeks, 56 weeks, 58 weeks, 60 weeks, 62 weeks, 64 weeks, 66 weeks, 68 weeks or 70 weeks.

The immunisation regimes typically have from 1 to 6 administrations of the immunogenic composition, but may have as few as 1, 2, 3, 4 or 5. The methods of inducing an immune response can also include administration of an adjuvant with the immunogens. In some instances, annual, biannual or other long interval (5-10 years) booster immunisation can supplement the initial immunisation protocol.

A specific embodiment of the invention provides methods of inducing an immune response against HIV in a subject by administering an immunogenic composition of the invention, , one or more times to a subject wherein the peptic sequence is expressed at a level sufficient to induce a specific immune response in the subject. Such immunisations can be repeated multiple times at time intervals of at least 2, 4 or 6 weeks (or more) in accordance with a desired immunisation regime.

The immunogenic compositions of the invention can be administered alone, or can be co-administered, or sequentially administered, with other HIV immunogens and/or HIV immunogenic compositions, e.g., with ‘other’ immunological, antigenic or vaccine or therapeutic compositions thereby providing multivalent or ‘cocktail’ or combination compositions of the invention and methods of employing them. Again, the ingredients and manner (sequential or co-administration) of administration, as well as dosages can be determined taking into consideration such factors as the age, sex, weight, species and condition of the particular subject, and the route of administration.

When used in combination, the other HIV immunogens can be administered at the same time or at different times as part of an overall immunisation regime, e.g., as part of a prime-boost regimen or other immunisation protocol.

Many other HIV immunogens are known in the art. One such immunogen is HIVA (described in WO 01/47955), which can be administered as a protein, on a plasmid (e.g., pTHr.HIVA) or in a viral vector (e.g., MVA.HIVA). Another such HIV immunogen is RENTA (described in PCT/US2004/037699), which can also be administered as a protein, on a plasmid (e.g., pTHr.RENTA) or in a viral vector (e.g., MVA.RENTA).

For example, one method of inducing an immune response against HIV in a human subject comprises administering at least one priming dose of an HIV immunogen and at least one boosting dose of an HIV immunogen, wherein the immunogen in each dose can be the same or different, provided that at least one of the immunogens is an epitope of the present invention, a nucleic acid encoding an epitope of the invention or an expression vector, preferably a VSV vector, encoding an epitope of the invention, and wherein the immunogens are administered in an amount or expressed at a level sufficient to induce an HIV-specific immune response in the subject. The HIV-specific immune response can include an HIV-specific T-cell immune response or an HIV-specific B-cell immune response. Such immunisations can be done at intervals, preferably of at least 2-6 or more weeks.

‘Treatment’ includes both therapeutic treatment and prophylactic or preventative treatment, wherein the object is to prevent or slow down the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. The terms ‘therapy’, ’therapeutic’, ‘treatment’ or ‘treating’ include reducing, alleviating or inhibiting or eliminating the symptoms or progress of a disease, as well as treatment intended to reduce, alleviate, inhibit or eliminate said symptoms or progress. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, methods and compositions of the invention are used to delay development of a disease or disorder or to slow the progression of a disease or disorder.

Preferably, an effective amount, preferably a therapeutically effective amount of the (poly)peptide or vector of the invention is administered. An ‘effective amount’ refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. The effective amount may vary according to the drug or prodrug with which the (poly) peptide or vector is co-administered.

A ‘therapeutically effective amount’ of a (poly) peptide or vector of the invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the (poly) peptide, to elicit a desired therapeutic result. A therapeutically effective amount encompasses an amount in which any toxic or detrimental effects of the (poly) peptide are outweighed by the therapeutically beneficial effects. A therapeutically effective amount also encompasses an amount sufficient to confer benefit, e.g., clinical benefit.

Throughout the specification, terms such as ‘comprises’, ‘comprised’, ‘comprising’ and can have the meaning attributed to them in most patent jurisdictions, preferably in the jurisdiction in question; e.g. they can mean ‘includes’, ‘included’, ‘including’, etc. Terms such as ‘consisting of ‘consisting essentially of and ‘consists essentially of have the meaning ascribed to them in most patent jurisdictions, preferably in the jurisdiction in question; e.g., they may imply the exclusion of all, most, or all but a negligible amount of other elements, or they may allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

An ‘isolated’ peptide or nucleic acid may be isolated substantially or completely away from one or more elements with which is associated in nature, such as other naturally occurring peptide or nucleic acids or other peptide or nucleic acid sequences. The invention will now be described in more detail by means of the following non limiting figures and examples. All patent and literature references cited herein are hereby incorporated by reference in their entirety.

FIGURES:

FIGURE 1. FabA 43 conformational epitope P7 designed in silico blocks FabA 43 binding to gp41 and reverse FabA 43 neutralization activity. A : Preincubation of FabA 43 with the conformational epitope P7 (5 mM, hatched bars) but not with control HA (5mM, plain bars) interferes significantly with FabA 43 binding to clade A, clade B and clade C PI or clades A and C gpl40 and clade B gp41, as evaluated by ELISA. B : The P7-induced reduction of FabA 43 binding to each antigen is dose dependent. Binding inhibition to FabA 43 binding to each antigen in the presence of P7 is calculated relative to the binding inhibition in the presence of the irrelevant HA peptide serving as negative control. In A and B, values represent mean ± SEM, derived from 3 independent experiments performed in triplicate. C : Localization of the amino acids paths corresponding to P7 peptide on the 6-Helix bundle conformation of clade A, B and C gp41 framel5. Each gp41 monomer of the trimer is depicted using a different tone of gray. The amino acid paths corresponding to the FabA43 specific peptide P7 are highlighted in orange. Note residues 532-535 belong to the N-Helix of one monomer while residues 669- 672 belong to the C-Helix of another monomer of the trimer. Red: the P7 predicted conformation superimposed on the structure of gp41. RMSD values over the paired residues of gp41 and P7 are of 2.45, 2.97 and 1.97 A for clade A, B and C, respectively. Note that in this region of the gp41, only residue 535 differs between clade B in one hand and clade A and C in the other hand. D : Conformational variability of the predicted conformations of P7 using PEP-FOLD3. Left: conformation best fitting gp41. Right: top 10 predicted conformations superimposed. E : Interference of FabA 43 neutralization by a 400-fold molar excess of P7 peptide. The percentage of neutralization, analyzed by flow cytometry as indicated in Materials and methods section, is shown relative to FabA 43 neutralization using primary CD4+ T cells in the presence of the irrelevant HA peptide, tested in parallel. Values represent mean ± SEM, derived from 3 independent experiments performed.

FIGURE 2: P7 neutralisation of HIV infection of CD4+T cells

P7 induce inhibition of CD4⁺T cells infection in a dose-dependent manner. The potential of P7 to inhibit CD4⁺T cells infection was assessed in a single cycle infectivity assay, using p24 staining on primary CD4⁺T cells. P7, at indicated concentrations, was preincubated for 1 h at 37° with either free virus (dotted line, diamond) or activated CD4⁺T cells (dotted line, triangle). After lh, activated CD4⁺T cells were added when P7 was preincubated with the virus, or virus was added when P7 was preincubated with CD4⁺T cells. Cultures were then incubated for 36 h. The percentage of neutralization, analyzed by flow cytometry, was defined as the reduction of the Gag-p24⁺ CD4⁺T cells in the presence of P7 compared with HIV- 1 -infected CD4⁺T cells in the absence of P7 (solid line, circle). Values represent mean ± SEM, derived from at least 4 independent experiments performed in triplicate.

FIGURE 3: P7 binds to trimeric envelope gpl40 cross clade

A. Binding of P7 (at increasing concentration up to 20 mM) to HIV-1 envelope was evaluated by ELISA after immobilization of gpl40 of either clade A (plain black bars), B (white bars) and C (hatched bars). B. The irrelevant 9 amino acid influenza virus Hemagglutinin (HA) peptide (YPYDVPDY) at 20 mM was used as a negative control. Peptide binding was detected with FabA 43 followed by HRP coupled anti-human IgA. n= 3 independent experiments. Student’s t-test, * = p<0.05 ***; = p<0.001

MATERIALS AND METHODS

- Proteins and Peptides

The gp41 (584-684) used was a construct based on the HXB2 group M subtype B sequence [40] and kindly provided by Dr. Weissenhorn. Gpl40 from HIV-1 clades A (92RW020) and C (C.ZA.1197MB) were obtained from Immune Technology Corp (NY10021, USA). Sequences of PI (a.a 630-685) derived from clade B HXB2 gp41 (Pl-B) was SQTQQEKNEQELEELDKWASLWNWFDITNWLWYIK (SEQ IDN°2) as described [22], from clade A 99UGA07072 gp41 (PI -A) was SQIQQKKNEQDLLALDKWANLWNWFDISNWLWYIR (SEQ IDN°3) and from Clade C Bw96Bw0502 (Pl-C) was SQTQQEKNEQELLALDS WKNLWNWF SITNWLWYIK (SEQ IDN°4). Peptides were chemically synthesized at a purity >95% by Biopeptide (LA, USA) for Pl-B and United BioSystems (VA, USA) for Pl-A and -C.

- Cloning of FabG and Production and Purification of soluble FabA and G

ESN Fab As were transformed in their corresponding FabGs by molecular cloning using pASK88 vector which direct the synthesis of the human gamma- 1 heavy chain and light chain, respectively [41] Production of functional Fabs was performed as previously described [2] Briefly, cultures were grown in 1L of LB medium containing 100pg/ml ampicillin and expression was induced for 14 hr at 22°C by addition of 0.2 mg/L of anhydrotetracycline (ACROS Chimica) at an OD550 of 0.5. Fabs were purified from the periplasmic fraction of the E. coli pellet by immobilized metal affinity chromatography (IMAC) using a GE Healthcare kit (GE Healthcare, Sweden, 17-5286-01, HisTrap FF crude). After elution from the affinity media with 0.5M imidazole, the FabA and G were dialyzed against PBS and concentrated using Amicon Ultra- 10 centrifugal filter units (Millipore). Antibody purity was evaluated by native SDS/PAGE, followed by Coomassie blue staining resulting in a single band at 50 kDa corresponding to the Fab heterodimer comprising the heavy and light chains, as expected. No band at 25 kDa corresponding to either isolated Fab light or heavy chain could be detected.

The concentration of FabA and G was measured by sandwich ELISA allowing the restricted detection of full Fabs, namely covalently linked heterodimers, using goat anti human IgA or IgG (Caltag, France) for coating, and biotinylated mouse anti-human Ig kappa light chains (B.D. Pharmingen, USA) for detection, as earlier described [2]

- Binding assays

Surface Plasmon Resonance (SPR): All experiments were performed in duplicate, as described in [11, 42] with a Biacore 3000 instrument (Biacore, Inc.) at 20°C in HBS-EP running buffer [10 mM Hepes (pH 7.4), 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20] Immobilization of various HIV-1 envelope subunit ligans ligands, namely Clade B gp 41, Clade A and C gpl40, and clade A, B and C PI peptides, to CM5 chips (GE Healthcare) followed the standard procedures recommended by manufacturer. The final immobilization levels were between 300 and 500 RU to avoid rebinding events, as mentioned in [42] Initial binding experiments with clone 43 and 177 of FabA and G, and non-specific isotypes as analytes were comparatively performed. No specific signal was observed with non-specific isotypes (recorded value of 0 RU), indicating that the observed binding of Fabs the different HIV-1 envelope subunits was specific. For kinetic measurements, sensorgrams were obtained by passing various concentrations of the analytes at various concentrations as indicated, over the ligand surface, at a flow rate of 50pl/min, with a 3-min association phase and a 6-min dissociation phase. The sensor surface was regenerated between each experiment with a single injection of 35 mM NaOH and 1.3 M NaCl at a flow rate of 50 mΐ/min for 30sec. Identical injections over blank surfaces ran in parallel (and giving a value of 0 RU) were subtracted from the data for kinetic analysis. Binding kinetics were evaluated by linearization using “fit kinetics Langmuir binding type” with BiaEvaluation software (BiacoreTM). Relative Pearson's chi- squared tests assessing goodness of fit were always below 10, which indicated that the models used for fitting adequately describe recorded data.

ELISA: ELISA binding assays were performed as described [11] by coating microtiter plates (NUNC-Immuno Plate MaxiSorp Surface, or Peptide Immobilizer Exiqon Peptide Immobilizer, Exiqon) with gpl40 (trimeric rgp41 at 0.25 microg/well), PI (0.1 pg/well), peptides corresponding to conformational epitopes (0.1 pg/well), overnight at 4°C in PBS. Fab binding was detected with a biotinylated mouse anti-human Ig kappa light chains (BD Pharmingen) and streptavidin- HRP or with anti-human IgA HRP (Jackson ImmunoRe search) for the peptides corresponding to conformational epitopes. All experiments were performed with Fabs from at least three independent purifications, each in duplicate. For competition ELISA, Fab 43 was preincubated ON at 4°C with various concentration of P7 or the irrelevant Hemagglutinin (HA) (YPYDVPDY) peptide serving as negative control and added to gp41 clade B, gpl40 (clades A, C) or PI (clades A, B, C) coated on the ELISA plate at a final concentration of 0.8nM for FabA 43 and 0.05 to 1 OmM for P7. Fab binding to each gp41 subunit was finally detected enzymatically by using anti human IgA coupled with HRP.

Fab binding to HIV-1 infected cells: Binding assays were performed as described [43] For detection of Fab binding to native HIV-1 envelope at the surface of HIV-1 infected cells, FabA or G (5pg/ml) were incubated with 105 cells overnight at 4°C. To allow for direct isotype comparison, FabA and G were detected in parallel using the same fluorescein isothiocyanate (FITC)-conjugated mouse anti-human kappa light chain (BD Biosciences, San Jose CA, USA), 30 minutes at 4°C. Cells were next fixed with 4% paraformaldehyde, and were further stained for intracellular Gag-p24 with the phycoerythrin (PE) coupled anti-Gag KC57 murine monoclonal antibody (Beckman Coulter GmbH). Fab binding to target cells was quantified by flow cytometry using a Guava EasyCyte flow cytometer (Merck-Milipore), and analyzed using the dedicated InCyte software.

- Target cells

Peripheral blood samples from healthy donors, obtained from the Etablissement Franqais de Sang (Paris, France) were depleted of CD8+T cells with Rosette Sep cocktail, (StemCell Technologies Inc., France) and peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque. After stimulation for 2 days with 5pg/ml phytohemagglutinin (Sigma-Aldrich, St.Louis, MO) as described [11], CD8-depleted PBMCs were used for infection and neutralization experiments. Alternatively, CD4+T CEM-NKR lymphocytic cells (NK-resistant) expressing CCR5 (AIDS Research and Reference Program, NIH) we used as when indicated. To prepare CD4+ T target cells for neutralization assays, cells were split 1 :3 on the day of passage and used the following day.

Primary monocytes were purified from peripheral blood samples from healthy donors using human monocyte enrichment kits (StemCell Technologies Inc., France) and differentiate into Langerhans cell with TGF-b, GM-CSF and IL-4, as described [11]

- Virus stock preparation

A stock of HIV-1JR-CSF (clade B, R5 tropic) was prepared on a large scale by transfecting 293T cells with a plasmid containing the DNA sequence of JR-CSF (NIH, Germantown, MD USA) [2] The cell culture supernatant was concentrated, separated into single use aliquots and stored at -80 deg C.

The HIV-1 primary isolates 92UG031 (clade A, R5) and 93BR025 (clade C, R5), obtained through the NIH AIDS Reagent Program, was amplified on PBMCs, as previously described [2] Virus concentration was quantified by measuring p24 antigen by ELISA (Innotest HIV-1 Antigen mAh, Innogenetics).

- HIV-1 Neutralization Assays

Single-cycle neutralization assay: The neutralization activity of FabA and FabG was evaluated on primary CD4+ T-cells (CD8+T cells-depleted PBMC), on CEM-CCR5+ infected with HIV-1 JR-CSF (Clade B) or with each of Clade A or C primary isolates and quantified by flow cytometry after intracellular Gag-p24 staining, as we described earlier [2] At least five independent experiments, performed each in triplicate, were performed. Live cells initially gated by forward and side scatter were analyzed for intracellular expression of p24-Ag. A dose dependent parameter was used to compare the FabA with FabG and for determination of maximum percent inhibition values. Neutralization was defined in % of cells infected in the absence of antibody. Titers were calculated as IC50 and IC85 and reported as the concentration of Fab causing a 50 or 80% reduction in the percentage of p24+ cells compared to virus control wells.

For competing the neutralization, FabA 43 was preincubated with a 400-fold molar excess of P7 overnight at 4°C and further incubated the virus and the target primary CD4+ T-cells at final FabA 43 concentration of 20nM and P7 concentration of 33 mM. The irrelevant peptide from the Hemagglutinin HA (YPYDVPDY) serving as negative control had no effect on FabA 43 neutralizing activities.

Inhibition of HIV-1 transfers from cells to cells. The inhibition of HIV-1 transfer from Langerhans cells to T cells was evaluated using monocytes derived Langerhans cells (LCs) obtained from PBMCs, and autologous TCD4+ cells as we previously described [11] Briefly, LCs were incubated with either clade of HIV-1 for 2hrs at 37°C, washed extensively to remove the free virus and distributed in 96 well plates at 100,000 cells / well. Indicated concentrations of FabA or G were then added to the corresponding wells. Finally, either medium alone, or resting TCD4+ cells in medium (100,000 cells / well) were added. The LCs-T co-cultures were incubated at 37°C for 5 days. The virus transfer was evaluated by measuring Gag-p24 released in the culture medium using a commercial ELISA (Innotest HIV-1 Antigen mAb, Innogenetics) according to manufacturer instructions. Results are expressed as % of inhibition transfer using formula [(LC+T-Ab) - (LC+T+Ab) / (LC+T-Ab)] xlOO.

- Epitope mapping

Epitope mapping of both isotypes was performed as we described previously [11], using linear 12-mer peptide libraries displayed on the protein pill of M13 phages (New England Biolabs) as recommended by the manufacturer. In brief, IgA- or IgG-coated beads were incubated with Fab IgA or IgG on a rotating wheel for 2 h at room temperature and epitope screening was initiated by incubating each bead of Fab IgA or IgG with the original 12-mer (1013) phage displayed peptide library containing different phages, overnight at 4°C. After three rounds of selection with increasing stringency, the phages were tittered, and single clones were picked and tested by phage ELISA for specific binding on each FabA or FabG. Positive clones were amplified, and each specific peptide insert was sequenced. Importantly, two round of negative selection using beads coated with normal human IgA or IgG (Jackson ImmunoRe search) were introduced between two rounds of positive selections. Phages remaining from the negative selection were amplified in Escherichia coli ER2738, precipitated, and used for second and third rounds of selection similar to the first round, but with increased buffer stringency as described [11]

- Modeling clade A, B and C gp41

Six Helix bundle early post fusion clade B gp41: X-ray crystallography studies have been highly successful in giving information about the helical regions of the HIV-1 gp41 ectodomain in its 6-Helix bundle state; however, in all cases to date the loop region was either removed to facilitate crystallization or not visible in the crystal structure [44]; [33]). However, the structure of the SIV gp41 ectodomain containing the loop region was successfully determined by NMR spectroscopy [45] The HIV-1 and SIV loops share a high degree of sequence identity (46%). The structure of the trimeric gp41 ectodomain of HIV-1 in its 6-Helix bundle state was thus built using the X-ray crystallography data available for the six-helix bundle of HIV- 1, and the NMR data available for the loop of SIV using the MODELLER v9.15 software [46] The conserved disulfide bond of the loop between residues C68 (C599) and C74 (C605) was added as a constraint. This bond was proved to be critical to the furin recognition site of HIV- 1 gpl60 [47]

The crystal structure of the 6-Helix bundle HIV-1 gp41 including both fusion peptide and membrane proximal external regions [33] was retrieved from the RCSB Protein Data Bank (http://www.rcsb.org, code : 2X7R). The solution NMR structure of ectodomain of SIV gp41 [48] was retrieved from the RCSB Protein Data Bank (http://www.rcsb.org, code : 2EZO). Multiple sequence alignment was used to build the 6- Helix bundle structure of HIV- 1 gp41.

Pre fusion clade B gp41: The structure of the trimeric gp41 ectodomain of HIV- 1 in its pre-fusion state was built using the sole crystallographic structure of the pre-fusion HIV-1 gp41 and missing loops were modeled using the BCLoopSearch algorithm (http://bioserv.rpbs.univ-paris-diderot.fr/services/BCSearch/) which helps identify linear fragments similar to a query in large collections of structures using a new similarity approach based on a Binet Cauchy (BC) kernel [49]

The crystal structure of the pre-fusion HIV-1 gp41 [32] was retrieved from the RCSB Protein Data Bank (http://www.rcsb.org, code : 4TVP).

Pre and post fusion Clade A and C gp41: The clade A and clade C gp41 sequences were retrieved from http://www.hiv.lanl.gov (code : AF484478 and code : AF110967, respectively) aligned and mapped onto the previous structures using the MODELLER v9.15 software [46] We used all atom molecular dynamics (MD) simulations to inspect the behavior of the 6 trimeric structures (pre-fusion : clade A, B and C, 6-Helix bundle : clade A, B and C) and explore their conformational space. The simulations were performed under periodic boundary conditions by defining a box with a minimal distance of 1.0 angstroms between the protein and the wall of the cell. The box was then filled with water and we added a concentration of 0,2 M Na+Cl- to the solvent, as well as an excess of Na+ ions to neutralize the system charge induced by the negatively charged residues. 200 ns of Molecular Dynamics simulations were then run for the structures using Gromacs 2016.1 [50] [51] with AMBER99SB-ILDN [52] as force field and TIP3P [53] model for water.

- Mimotope Sequence Analyses and docking on gp41

For each set of Fab A and FabG mimotopes, epitope mapping on pre and post fusion clade A, B and C gp41 was performed by computational method that aims to predict discontinuous or 3D eptitopes. Three available computational methods to map mimotopes sequences to conformational epitopes were evaluated: PepSurf, Mapitope [54] and [55] We selected PepSurf for this study because it provided the best results - as it was able to retrieve the PI epitope on both pre-fusion and 6-Helix bundle structures using mimotopes of 2F5 IgG and IgA - and allowed the user to define the similarity matrix. A discontinuous 3D epitope is localized on the protein surface by searching for a 3D fit with partial amino acid strings of a given sequence in a preset distance. PepSurf compares each peptide provided to the solvent accessible surface of the antigen, determining the best path. The algorithm then creates clusters of antigen residues on the surface that best fit a grouping of peptides and returns a score for each residue at the surface corresponding to the number of occurrences of this residue in the alignments.

Snapshots of the gp41 trimeric structures were taken every 10 ns of the MD starting from 60 ns and explored using the pepSurf algorithm with both BLOSUM62 and BLOSUM80 as similarity matrix and the 141 antibody binding mimotopes as input. A library of 147 12-mer soluble peptides was generated using SolyPep on the RPBS web portal and used also as input to account for the background noise. Scores were then averaged for each residue position for the three chains of the 15 structure snapshots to account for the protein flexibility. Scores obtained for the random peptides library were subtracted.

Using this method, we found three regions that could correspond to conformational 3D epitopes on the 3 6-Helix bundle structures:

• G1-Q10 and E/A132-F143 which correspond to the N-terminus of the first helix and the C-terminus of the second helix of each monomer, respectively,

• E30-T39 and 1105-1/Ll 16 which are roughly located in the middle of both helices,

• Q60-W66 and A/N77-A/S83 which encompass the top of the helices and the loops. Results for the 3 pre-fusion structures were less clear but highlighted a region in the vicinity of the PI epitope (A/E132-F143) and a region near the epitope found in the 6- Helix bundle structure at A/N77-A/S83.

Although PepSurf consider the relative distance and physicochemical properties of individual amino acids, it does not take into account the 3D conformation that the mimotopes might adopt in the solvent. The 141 tested mimotopes that encompass the found epitopes were thus modeled using the PEP-FOLD3 algorithm, a de novo approach aimed at predicting peptide structures from amino acid sequences, based on structural alphabet letters [56] The peptide structures were then aligned to the paths found by the PepSurf algorithm on all 6 structures of gp41 (pre-fusion : clade A, B and C, 6-Helix bundle : clade A, B and C) by a pair-fit procedure to segregate paths that comply with the 3D conformation. Conformational paths - that is, paths that encompass two different chains - were selected and the resulting sequence modeled with the PEP-FOLD3 algorithm. Only peptide structures that aligned to the same sequence in the gp41 structure with a RMSD lower than 2,5 angstroms were retained, in order to keep only the peptide that would fold in water in the same manner as in the gp41 structures.

- Statistical analysis

Statistical significance was analyzed by the two-tailed Student’s t-test and multiple comparisons were performed using the non-parametric Mann- Whitney U test using Prism 5 (GraphPad, San Diego, CA) software. Heatmap was established using the heatmaper web site (http://www.heatmapper.ca/expression/) using the complete linkage clustering method and non-parametric spearman correlations as distance measurement method. P values <0.05 were considered significant.

RESULTS

1.1. CHI Isotype switching of mucosal FabA into FabG

To evaluate the role of the CHI domain in mucosal Ab specificity and function, we took advantage of the mucosal anti-HIV-1 FabA derived from cervical B cells of ESN individuals we previously characterized [2] Two FabA clones, namely 43 and 177, were obtained after screening of the ESN library on the clade B gp41 membrane proximal extended region (MPER) PI [18] [19] or using a recombinant Clade B gp41 deleted for its MPER [2], respectively. At the molecular level, FabA 43 has a high degree of somatic mutations, a VH3 heavy chain origin similar to the broadly neutralizing IgG 4E10 [20], and a long CDRH3 of 22 residues, features characteristics of other broadly neutralizing IgGs [2] In contrast, FabA 177 has a low level of somatic mutations for their heavy chain, 100% homology with germ-line gene region IGKVID-30*-01, and a normal length CDRH3 of 11 residues [2] Furthermore, FabA 177 heavy chain origin is VH6 family, which was never described before for broadly neutralizing IgGs. Each FabA was transformed by genetic engineering replacing the CHlal into CHlyl , as described in the method section, in their corresponding FabG, the two isotypes thus sharing identical VH and VL domains and the same light chain, but expressing different CHI domain.

1.2. FabA and FabG binding and affinity for HIV-1 envelope

We first evaluated whether class- switching preserves the original binding properties of IgA by comparing FabA and FabG binding to recombinant trimeric gp41 derived from HIV-1 Clade B [19] and gpl40 derived from HIV-1 Clades A and C. To avoid bias introduced by different isotype specific detection antibody, binding to gp41 in ELISA was detected using mouse anti-human kappa light chain [11] FabA 43 and 177 recognize conformational conserved regions on clade B gp41 from both R5 and X4 tropic viruses [2] Here, we found that both FabA and G pairs specifically bind to Clade B gp41 as well as to Clade A and C gpl40 in a dose-dependent manner, but strikingly for all clades, binding of FabA 43 and 177 is more efficient compared to their respective FabG Hence, a 50 times higher concentration of FabG 43 (50pg/ml) is required to reach the same binding of FabA 43 (lpg/ml)). Differences observed between FabA and G 177 is even larger, reaching 500 between the isotypes in favor of FabA. Similarly, binding to clade A and C gpl40 requireslO times less FabA 43 than FabG 43 and 50-100 times less FabA 177 than FabG 177. Importantly, these results show that both ENS clones recognize the viral envelope cross clade.

Surface plasmon resonance (SPR) was next used in kinetic experiments to compare of FabA and G affinity to clade B gp41 or clade A or C gpl40 immobilized onto the sensor ship surface. FabA and G 43 and 177 at various concentrations were the analytes. As a result (Table 1), when Clade B gp41 serves as target, KD is 3.62 nM for FabA 43 and 21.2nM for FabA 177 compared to 1.12 and 1.22 mM for their respective FabG. When Clade A and C gpl40 serve as target, KDs are 6.41 and 4.79nM for the FabA 43 compared to 0.50mM and no affinity is measured for its respective FabG; KD equals 2.92 and 2.15nM for FabA 177 whereas its corresponding FabG has no measurable affinity for any of the clade A or C viral envelope. Overall, affinities reach the nM range for the FabA, whereas remain in the order of mM or even lower for the FabG for both clones, in agreement with binding experiments using ELISA and show that the affinity of both FabA 43 and 177 are greater, for all clades, than that of their FabG counterpart. Importantly, for all Fab taken together, affinity correlates with binding observed by ELISA (spearman r= 0.606; p=0.375)

Altogether, FabA 43 and 177 bind to clade B gp41 and clade A and C gpl40 with nM affinity whereas affinities of corresponding FabG are much lower, although still able to bind their targets as shown by ELISA approaches.

1.3. Binding of FabA and FabG to the PI peptide

The 35 amino acid peptide PI was characterized as the minimal membrane proximal external region of the viral envelope gp41 that allows for HIV-1 binding to galactosyl ceramide, the HIV-1 mucosal receptor and for mucosal HIV-1 entry by transcytosis [21] [22] [23] This highly conserved region has been used as immunogen in a prophylactic vaccine against HIV-1 both in pre-clinical and clinical phase I trials [19, 24] PI sequence is well conserved between clade B and A viruses whereas a K670S mutation in clade C virus prevents binding of the broadly neutralizing 2F5 IgG [25] FabA 43 was obtained by screening the FabA ESN library on clade B PI and consequently FabA 43 binds to clade B PI [2] We thus evaluated by ELISA the capacity of FabA and G 43 to bind to clade A, B and C PI, comparatively as described [2] Both Fab 43 isotypes bind to clade A, B and C PI in a dose-dependent manner. Strikinly, as above when the PI peptide is expressed in the context of a full protein, FabA 43 recognizes PI more efficiently than its corresponding FabG. This difference is higher when clade B PI is the target (with a 60- fold change from FabA to G), but still significant (with a 20-fold change from FabA to G) when Clade A and C are targets. Additionally, FabA and G 43 bind more efficiently to Clade B compared to Clade A and C PI. These differences in Fab isotype binding to PI from the three clades are also observed by SPR.

Overall, the FabA 43 and 177 affinities for the gp41 viral envelope cross clade is in the nM range, similar to that of other broadly neutralizing Abs (bNAbs) [26] whereas FabG recognize less efficiently the same antigen.

1.4. FabA and FabG binding to HIV-infected CD4⁺ T-cells

We next evaluated the capacity of Fabs to bind to the viral envelope in its trimeric spike conformation at the surface of CD4⁺ T-cells infected with clade A (primary isolate 92UG031), B (isolate JR-CSF) or C virus (primary isolate 92BR025). After overnight incubation at 4°C with the various Fabs, binding was evaluated by flow cytometry using an anti-kappa light chain secondary antibody to allow direct isotype comparison. All Fabs bind specifically HIV-1 infected cells although recognition of Clade C and clade A HIV-1 infected cells by FabG 43 and FabG 177 is low. Irrelevant IgA and IgG used as negative controls give negligible binding signal. As observed for recombinant viral protein binding, for all clades, FabA 43 and 177 bind HIV-1 infected cells better than their respective FabG with 20-40% of T-cells labelled for FabA compared to 5-19% for FabG (Student’s t -test, p<0.05) for Fab 43 and 177 respectively). FabA 43 recognizes more efficiently Clade A than clade B and C infected CD4⁺ T-cells (Student’s t -test, p<0.05), whereas no differences were apparent between clades for FabA 177. Importantly, Fab binding to HIV- 1 infected cells correlates with Fab affinity and binding to HIV-1 envelope measured by ELISA (spearman r= 0.777 p=0.0156).

1.5. FabA and FabG antiviral properties

As FabA and G only differ by the CHI domain, the results presented above suggest that the CHI domain can affect the paratope and its fitting on the antigen and may in turn impact the Ab anti-viral function in an isotype-dependent manner.

- Neutralization of HIV-1 CD4⁺ T-cell infection by FabA and FabG

We first investigated the participation of the CHI domain in Fab isotype neutralizing activities, comparatively. Using a panel of three viruses from Clade A, B and C, neutralization of infection by FabA and G pairs of both 43 and 177 clones of CD4⁺T lymphocyte was tested, as previously described [2] Serial dilutions of each FabA and G were assessed comparatively, and IC50 values were determined for each HIV-1 clade. Comparative evaluation of paired Fab 43 isotypes reveals that FabA43 neutralizes all three A, B and C clades with respective IC50 of lng, lpg and lOOng/ml, whereas FabG 43 only neutralizes clade A virus with an IC50 of lOOng/ml and not Clade B or C virus clades. A similar neutralization pattern is observed for the clone 177 with an IC50 of lng, 300ng and 30ng/ml for FabA 177 against Clade A, B and C virus respectively and an IC50 of lOng and lpg for FabG 177 against Clade A and C virus respectively. In all cases, the paired FabA has significantly increased neutralization compared to FabG for all concentrations evaluated (Student’s t -test, p values ranging from p<0.01 to p<0.00001).

In summary, for all three HIV-1 clades, the CHla provides FabA with the capacity to neutralize CD4⁺ T-cell infection whereas the corresponding FabG harboring the same paratope but a CHly has limited neutralizing activities.

- Blockade of cell-to-cell virus transfer by FabA and FabG

Cell-to-cell transmission of HIV-1 is of major importance in vivo , being much more efficient than infection by cell-free virions [27] At the mucosal level, HIV-1 entry through multilayer mucosal tissues occurs mainly by targeting Langerhans cells (LCs) that in turn transfer to CD4⁺ T-cells [28] [29] [30] [31] that constitute the founder infected population. Moreover, CD4⁺T cells can disseminate out of the mucosal tissue and spread the virus to other CD4⁺T cells. Thus, we evaluated the capacity of the two pairs of ESN FabA and G to block clades A, B and C virus transfer from LCs to CD4⁺T-cells. Transfer of clade A and C HIV-1 is inhibited by both FabA 43 and 177 in a concentration dependent manner, as well as that of clade B by FabA 43, inducing a >40% inhibition at lOOng/ml. Transfer of clade B virus remains much less sensitive to FabA 177 . FabG 43 blocks transfer of Clade A and C viruses but less efficiently than its FabA counterpart and FabG 177 was only able to block clade A HIV-1 transfer. Of note, taken all the value together (including all viral clades and Fab isotypes) at lOOng/ml of Fab, neutralization activities correlated directly with inhibition of virus transfer (spearman r= 0,6655, p= 0,0219).

Taken together, this set of functional analyses demonstrate that switching the CHla from a mucosal Ab to CHly considerably reduces the Fab antiviral properties, either neutralization or HIV-1 transfer from LCs to CD4⁺T-cells.

1.6. FabA and G epitope mapping

As a further step in elucidating the molecular bases by which each specific CHI region impact on antibody affinity and functions, due to selective molecular stringency/flexibility imposed by CHI domain on the paratope conformation, we thought to determine the conformational epitopes specific for each Fab isotype. The molecular bases by which each specific CHI region impact on antibody affinity and functions could be due to selective molecular stringency/flexibility imposed by CHI domain on the paratope conformation. Thus, we thought to determine the conformational epitopes specific for each Fab isotype. Clones 43 and 177 specific FabA and FabG epitopes were characterized by combining epitope mapping, by screening a random peptide library, with an in-silico analysis. For this later, we used available clade B gp41 crystal structures, either in pre- or 6-Helix bundle conformations, that were further adapted by in silico modeling to model clade A and C gp41, as described in the method section.

Therefore, a 12mer random peptide library was used to characterize a set of the best specific peptides, referred to as mimotopes, for both 43 and 177 clones as FabA and FabG using three rounds of successive screening with increasing stringency, as we described [2]

After sequencing 100 phages specific for each of the Fabs, a larger set of peptides with higher occurrence were retrieved on each of the FabA compared to FabG, in line with the lower affinity of the FabGs compared with Fab As for gp41. We therefore concentrated our analysis below on the FabA 43 and 177 mimotopes. None of the selected mimotopes correspond to linear sequences of gp41, indicating that Fab epitopes are conformational, as already suggested [2]

To characterize these conformational epitopes, each set of FabA mimotopes was docked onto trimeric gp41 crystal structures using in silico approaches, as described in the method section and supplementary data. Only two gp41 crystal structures of a Clade B gp41 in different conformational states are available. One structure represents gp41 in the pre-fusion state together with gpl20, although this gp41 lacks the MPER region [32] The other gp41 structure mimics the post fusion state and is composed of three N-helices and three C-helices that form a six helix-bundle for completion of HIV- 1 fusion with target cells in a spring load model of fusion. In this case, the gp41 construct lacks the gp41 Cys- loop bridging the C and N helices [33]

Clone 43 is primarily specific for the gp41 MPER [2] and the Cys-loop contains important gp41 epitopes [34] Therefore, we first optimized the available crystal structure of gp41 clade B by reconstituting the missing MPER in the pre-fusion structure and the missing loop in the 6-Helix bundle as described in the supplementary data. As no crystal structure of clade A or C are accessible in the literature, we constructed A and clade C gp41 structure by mapping each of clade A and Clade C gp41 sequences onto the clade B gp41 structures, as detailed in the supplementary data. As a result, from these in silico modeling calculations, Clade A, B and C gp41 structures in the pre- and 6-Helix bundle conformation were available for epitope docking studies.

Selected FabA specific mimotopes obtained from FabA 43 and 177 were localized on clade A, B and C gp41 pre-fusion and six-helix bundle structures using the Pepsurf method as detailed in supplementary data. Three main regions were targeted by FabA 43 mimotopes on gp41 helix bundle structure of all three clades the highest hits being localized on the lower MPER region interface with the N-helix, in agreement with the screening strategy focusing on PI used for selecting FabA 43 [2] In comparison, FabA 43 mimotopes also fit on the pre-fusion structures, although in a more scattered manner and with lower scores. The main regions targeted by FabA 177 mimotopes on gp41 helix bundle structure of all three clades differ, as expected from the FabA 177 screening strategy, from those targeted by FabA 43 mimotopes, and the best fit appears in the loop linking the N and C gp41-helices. These FabA 177 mimotopes also match gp41 pre-fusion structures, but with lower scores and in a more scattered pattern, irrespective of the clade.

From these analysis, for each gp41 conformation and clade, an additional bioinformatic analysis described in the method section, allowed to characterized for FabA 43, 5 conformational epitopes that best mimic each set of mimotopes that could be used as a vaccine. Interestingly, only one of this conformational peptide was extracted from gp41 in a pre-fusion conformation, in agreement with lower scores obtained for mimotopes docking on pre-fusion versus 6-Helix bundle gp41 structure as shown above.

One out of 5 conformational epitopes defined in silico for FabA 43, referred to as P7, competed significantly with FabA 43 binding to PI and gp41/gpl40 of the three clades in ELISA when preincubated with the FabA 43 (Figure 1A) in a concentration-dependent manner (Figure IB). P7 at 5 mM could block FabA 43 binding by >50%, reaching >80% at 1 OmM (Figure IB). A 9 aa peptide derived from the influenza virus hemagglutinin (HA) used as negative control did not interfere with 177 binding in ELISA (Figure 1 A).

In contrast, peptide P7 did not interfere with Fab G 43 binding to the same antigens (not shown). Altogether, these results indicate that P7 interfere with FabA 43 cross-clade. P7 corresponds to an 11 aminoacid peptide located at the interface formed by the tips of Isl and H-helices of the three clades of gp41 (Figure 1C). This cross-clade activity is unexpected as P7 has been defined as the best conformational epitope extracted from in silico on FabA 43 mimotope fitting to the 6-Helix bundle Clade A gp41 structure. The other four conformational peptides defined in silico for FabA 43 that do not interfere with Fab A 43 binding to the gp41 antigens are located on different regions of gp41 on each gp41 clade, although for two of them spatially close from the region defined by.

Furthermore, at the functional level, preincubation of Fab A 43 with a 400- fold molar excess of P7 blocked the FabA 43 HIV-1 clade A, B and C neutralizing activities with an efficacy of 33+5, 54+8, 24+11 %, respectively (Figure IE). These results are in good agreement with the antigen binding blockade observed in ELISA (Figure 1 A, B).

The potential of P7 to inhibit CD4⁺T cells infection was then assessed in a single cycle infectivity assay. P7 induce inhibition of CD4⁺T cells infection in a dose-dependent manner (Figure 2). Finally we evaluate whether P7 could bind to HIV-1 envelope trimers gpl40 of clades A, B and C. Therefore, P7 at various concentrations (5, 10 and 20mM) or irrelevant HA peptide (20mM) were incubated with each immobilized gpl40. P7 binding was quantified using FabA 43 for detection (Figure 3A-B).

Results indicate that FabA 43 binds to each gpl40 clade in the absence (0 mM) and in the presence of 5 mM of P7. However, in the presence of 20 mM of P7, but not of HA peptide (not shown), Fab43 binding to each gpl40 clade increases by a factor 2. P7 being the specific epitope of FabA 43 and thus binding to it, as we already demonstrated (Khamassi et al, Plos Parth 2020), these results suggest a mechanism by which P7 binds to gpl40 increasing the epitope number accessible to the FabA. Furthermore, these results show that P7 binds the viral envelope cross clade, as does FabA 43 (Figure 3A-B).

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Claims

1. An isolated peptide comprising or consisting of the consecutive sequence of amino acids of sequence: LWNWFDISAASI (SEQ IDN°1).

2. A therapeutic composition comprising a peptide according to claim 1 for the preventive or curative treatment of an HIV infection.

3. A vaccine composition comprising a peptide according to claim 1, or a nucleic acid encoding said peptide.

4. A composition according to claim 2 or 3, which is suitable for mucosal administration.

5. A vaccine composition according to claim 3, further comprising an adjuvant.

6. A peptide of claim 1 or a composition of any one of claims 2 to 5 for use in the preventive treatment of HIV infection or AIDS.

7. A peptide of claim 1 or a composition of any one of claims 2 to 4 for use in a method of inducing a mucosal immune response to HIV-1.

8. A peptide according to claim 1, or a nucleic acid encoding said peptide, for use in a method of inducing a mucosal IgA response to HIV-1, wherein said peptide has the sequence SEQ ID N° 1.

9. A peptide according to claim 1, or a nucleic acid encoding said peptide, for use in a method of inducing a mucosal IgG response to HIV-1, wherein said peptide has the sequence SEQ ID N° 1.

10 An isolated nucleic acid encoding the amino acid sequence of a peptide according to claim 1.

11. A method of prevention or treatment of an HIV infection, comprising administration of a composition of any one of claims 2 to 5 to an individual in need thereof.